Heparin and heparan sulfate
Heparan sulfate is a member of the glycosaminoglycan family of polysaccharides. These glycosaminoglycans are linear polymeric carbohydrate chains that are the glycan part of proteoglycan molecules that are important structural and functional components of the extracellular matrix.
Heparan sulfate and heparin like compounds share a biosynthetic pathway, and are characterized by a repeating disaccharide units consisting of a 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) residue linked a-(1-4) to a uronic acid residue in a repeating fashion. The glucosamine can exist in the 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-deoxy-2-sulfamino-D-glucose (GlcNS) and 2-deoxy-2-sulfamino, 6-O-sulfate-D-glucose (GlcNS6S) forms. The uronic acid residue can be either .beta.-D-glucuronic acid (GlcA) or .alpha.-L-iduronic acid (IdoA), the latter of which can exist in the 2-O-sulfated form (IdoA2S). The exact composition of heparan sulfates varies significantly depending on the source and even the specific stage of cell growth. Heparin has been shown to be a more extensively transformed biosynthetic form of heparan sulfate. Heparin possesses a greater proportion of the IdoA2S and GlcNS6S residues and is therefore a more highly sulfated substance. In-vivo heparin is found in association with mast cells, and not with the extracellular matrix of vascularized tissue, as is heparan sulfate.
Heparin is a commonly employed anticoagulant and antithrombotic drug. Commercial heparin is usually isolated from porcine or bovine mucosa, or bovine lung tissue. The anticoagulant action of heparin has been shown to reside largely in its ability to interact with and potentiate the activity of a circulating protein, antithrombin III (AT-Ill). AT-III is a serine protease inhibitor that inhibits many of the serine proteases involved in the coagulation cascade, particularly thrombin (Factor IIa) and Factor Xa. Heparin interacts with AT-III to form a complex that inhibits thrombin and Xa much more effectively than AT-III alone. Heparin interacts with AT-III through a specific high affinity pentasaccharide sequence that represents only a minor portion of any heparin chain. Heparin also interacts with another serine protease inhibitor, heparin cofactor II, to potentiate the inhibition of thrombin. The interaction with HC-II operates through different structural requirements and does not appear to have the same degree of specificity as the AT-III interaction.
The potent anticoagulant related activities of heparin, unfortunately, preclude its use for many of the other potential indications. In particular, the anticoagulant activity of heparin can lead to hemorrhage and bleeding complications when administered at doses that yield other therapeutic benefit. This factor has led to an interest in selectively decoupling the anticoagulant activity of heparin from other biological properties of the molecule. This decoupling strategy can also be applied to selectively decoupling or retaining other desirable biological and therapeutic properties. More selective agents would have reduced risk of toxic and/or contra-indicative effects. Also, the specificity could enhance overall bioavailability and potency of the agent relative to heparin.
Heparin/heparan sulfate have been shown to interact with a large and expanding number of proteins, and have been associated with numerous biological functions and activities. This poly-pharmacy is mediated by the ability of heparin-like materials to bind and inhibit or stimulate the action of a myriad of extracellular matrix resident and circulating receptors, proteins and enzymes. The broad pharmacological action of heparin and related compounds are of considerable therapeutic interest for a large number of indications, particularly in cardiovascular related diseases. There is considerable precedent for the utility of heparin based therapeutics for disease states related to hemostasis and thrombosis, and a recently expanding interest in conditions related to atherosclerosis, angiogenesis, metastasis and inflammation.
There is a fairly large effort in developing heparin based therapies for treating thrombotic and proliferative vascular disorders. However, despite considerable in-vitro, and anecdotal in-vivo evidence there has been relatively less focus on the anti-inflammatory use of heparin derived materials. Some of the anti-inflammatory properties of heparin derived compounds include; inhibition of complement activation, inhibition of heparanase dependent T-lymphocyte migration, inhibition of leukocyte platelet interaction. In addition, heparin and derivatives have shown activity in animal models of DTH, EAE, ischemia reperfusion and asthma, as well as anecdotal description of benefit in humans suffering from asthma and ischemia (myocardial infarction).
As mentioned above, its potent anticoagulant activity makes unattractive its therapeutic use for cardiovascular disorders. In particular, the anticoagulant activity of heparin leads to hemorrhage and bleeding complications when administered in therapeutic doses.
Pathology of shock
In general, shock can be described as widespread hypoperfusion of cells and tissue due to reduction in blood volume or cardiac output or redistribution of blood resulting in an inadequate effective circulating volume. Shock is usually classified into tour types: (1) Cardiogenic; (2) Hypovolemic; (3) Septic and (4) Neurogenic, largely on the basis of the hemodynamic derangement that leads to the condition. Extensive study has resulted in an understanding of the general mechanisms that lead to the pathology associated with shock. However, the myriad of biochemical processes that mediate the pathology are only just beginning to be understood. This relates to a complex cascade of inflammation-related events that have been studied in association with hypovolemic and septic shock that result in toxic injury to cell membranes of endothelial and other organ cells, leukocytes and platelets, activation of intrinsic and extrinsic coagulation pathways, complement activation and formation of the vasoactive and chemotactic fragments C5a and C3a.
Hypovolemic shock, (2), and models for studying this condition are described by Chaudry and Ayala in "Immunological Aspects of Hemorrhage" (R. G. Landes Co., Austin, Tex., 1992). Generally, hypovolemic shock due to reduced blood flow associated with blood loss results in "sludging" of the blood and capillary "plugging" by erythrocytes, platelets and neutrophils. This in turn leads to the insufficient delivery of oxygen and nutrients to cells and tissues, deficient clearance of other metabolites, and to activation of neutrophils and platelets. This oxidant stress (Hypoxia) and release of other factors from the endothelium and macrophages stimulates the arachidonic acid cascade and the production of chemoattractant and inflammation mediators, leading to further neutrophil infiltration. The activation of neutrophils, platelets, macrophages and the complement cascade leads to the release of numerous biologically active agents including cytokines. These factors stimulate expression of adhesion molecules on the surface of the endothelium, neutrophils and leukocytes which permit binding and ultimately migration of the neutrophils and leukocytes through the extracellular matrix (ECM) and basement membrane of blood vessels and capillaries. This migration or extravasation is attributed to the action of a number of extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases and endoglycosidases (i.e.hcparanases), which are released by activated neutrophils, leukocytcs and/or platelets. The damage to the ECM and basement membrane results in increased vascular permeability, and infiltration of organs by neutrophils and leukocytes.
An analogous series of events is associated with septic shock except, and most critically, the key mediators of the inflammatory response are unlikely to be the same as those that cause hypovolemic shock. The initial blood volume reduction in septic shock occurs as a result of blood pooling after endotoxin stimulate neutrophil activation and the release of inflammation mediating cytokines (TNF, IL-1 and IL-6, IL- 10, TGF-.beta., etc. ).
It is important to keep in mind that hypovolemic and septic shock are distinct diseases. Hypovolemic shock is a general collapse of the circulatory system that can be caused by many events including any trauma to the circulatory system (e.g. gun shot wound, automobile injury, burns, stabbing, and so on). Septic shock, on the other hand, is caused by bacterial infection. Thus, as mentioned above, the causes of these diseases are highly likely to be distinct.
Ischemia/reperfusion injury (FRD is another instance where inflammation mediated cell and organ damage result after a reduced blood flow state (ischemia).
The vascular damage associated with hypovolemic shock, and the resulting infiltration of neutrophils and leukocytes into the various organs leads to tissue damage and ultimately multiple organ failure (MOF) and acute respiratory distress syndrome (ARDS). The destructive agents and mediators are numerous and include cytokines, enzymes and various other inflammatory agents. MOF and ARDS can occur in severe shock and often result in death. For therapeutic agents to be effective in shock, they must protect the microvasculature and various organs (liver, kidney, heart, spleen and gut) from failure. The importance of protecting or restoring gut function and intestinal function in hemorrhagic shock and I/R injury has been reported, and correlates with reduced septic complications, and long-term survival.
Heparin and Shock
Chaudry et al. (Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28) R645-R650, 1990 and J. of Trauma (1992) 32(4):420-426) have demonstrated that pre-heparinization can modulate the detrimental systemic effects that result from hypovolemic shock that can lead to MOF, and frequently death. This effect has been demonstrated in a rat hemorrhagic shock model (hypovolemic shock or traumatic shock) (Chaudry et al., Circ. Shock (1989) 27:318). This model differs from other hemorrhagic shock models mainly in that the animals are not heparinized prior to inducing shock, a situation that is more representative of the clinical situation. In this model, it was observed that pre-heparinized animals had significantly improved organ function relative to animals that were not heparinized but were otherwise subjected to hemorrhagic shock and resuscitated in the same manner. It was later shown that heparin administered during resuscitation had a similar beneficial effect, indicating that prior exposure to heparin was not necessary for protection.
The mechanism whereby heparin modulates the detrimental pathology of shock is not yet well understood. It is known that heparin and related heparan sulfate structures interact with a myriad of circulating proteins and enzymes, and a number of cell surface receptors. It may be that some of these interactions mediate shock damage. These biological activities and functions of heparin/heparan sulfate may depend on a number of chemical, compositional and physical characteristics, including: (1) saccharide composition; (2) functional group distribution; (3) charge density; (4) sulfate/carboxylate ratio; and (5) molecular weight. Regarding the latter, it has been demonstrated in the hemorrhagic shock model that the modulating effect of heparan is not directly related to the molecular weight of the heparin polymer, since a low molecular weight (LMW) heparin (average MW 3-4 kDa) had roughly equivalent effects. Chaudry et al., Circ. Shock (1991 ) 34:25).
In hypovolemic shock model studies with heparin that examined platelet function, it was concluded that the anticoagulant action of heparin may not be directly responsible for its action. Rana et al., J. of Trauma (1992) 32(4):420. This was supported by LMW heparin data, and by data obtained from a study using heparan sulfate, which has low anticoagulant activity, and which was found to be effective in preserving gut function after shock. Singh, et al., J. of Trauma (1993) 34:645.
Unfortunately, despite the work described above it is critical to keep in mind that none of these studies have elucidated the key structural requirements that a non-anticoagulant composition must have to be effective for treating or preventing hypovolemic shock.
Non-Anticoagulant (NAC) Heparin
Heparin's best known activity, its anticoagulant activity, prevents it from being used for a large number of possible clinical applications. Thus, considerable effort has been, and continues to be expended to identify heparin-like compounds for therapeutic use that have reduced anticoagulant activity and bleeding properties in vivo. Generally, two approaches have been pursued. The first being to identity forms of naturally occurring heparin that have lost anticoagulant activity. Included in this category are heparin fragments obtained by depolymerization and standard chromatographic separation methods, or by ATIII affinity fractionation. The second approach has been to chemically or enzymatically modify heparin to reduce its anticoagulant activity. In certain of these studies, heparin fragments may be prepared before or after chemical modification. Methods used for this latter approach include: (1) N-desulfation, N-modification; (2) N,O-desulfation and N-modification; (3) oversulfation; (4) carboxyl reduction or sulfation; and (5) periodate oxidation and reduction.
Not all of the above approaches to generating NAC heparins lead to compounds that fulfill the therapeutic criteria of low anticoagulant activity and limited effect on bleeding time when administered at therapeutically effective doses. Certain compositions may fulfill these criteria, but they also frequently lose the activity of interest. In other instances, the anticoagulant activity may be reduced, but the in vivo effect on bleeding remains elevated at useful doses. Some of these have been reported to be implicated in the inhibition of complement activation and C5a, in the inhibition of extracellular matrix degrading enzymes (elastasc, cathepsin G and heparanases), and some have been proposed as useful for treating shock. See for example, U.S. Pat. No. 4,916,219 which describes anticomplement pharmaceutical compositions with reduced anticoagulant activity. The compositions consist of small chain fragments of heparin produced by the depolymerization of heparin with heparinase of from about 6 saccharide units to about 24 saccharide units. Although no data are presented, it was suggested that such compositions could be useful for treating septic shock. There is no description of using the compositions for treating hypovolemic shock.
It is important to keep in mind regarding NAC heparins that, in most instances, the precise requirements for specific heparin-related activities are not known, and that the activity of modified heparins is not predictable.
An example of non-anticoagulant depolymerized low molecular weight heparin is described in U.S. Pat. No. 4,990,502. It shows the treatment of heparin with periodate, followed by depolymerization with base, and reduction of the aldehydes generated in the periodate treatment. The resulting material is said to contain a mixture of polymers containing 17-33 residues and containing a multiplicity of residues of the formula ##STR1## wherein the glucosamine residue is sulfated at the 3 and/or 6 position in an arbitrary manner, and wherein some of the IdoA residues may be replaced by cleaved IdoA or GlcA residues resulting from the periodate oxidation. These shortened polymeric chains are said to lack the binding site for ATIII but to be capable of inhibiting smooth muscle proliferation and to have physiological activities that include acceleration of tissue repair, prevention of atherogenous lesions, and prevention of the development of metastasis. Such compositions are also stated to be useful for the prevention of states of shock. There is, however, no discussion of using such compositions for treating hypovolemic shock.
Treatment of heparin/heparan sulfate with periodate has also been reported by others. For instance, Fransson, L. -A. and Lewis, W., FEBS Lett (1979) 97:1 19-123, describe a variety of conditions relating to the treatment of heparin/heparan sulfate with periodate and reduction by sodium borohydride or fragmentation in alkaline medium. Further, Fransson, L. -A. et al., Carbohydrate Res (1980) 80:131-145, studied the chemistry of various forms of heparin produced with periodate. In one study, the treatment with periodate was followed by .beta.-elimination in base to produce fragmentation. They further reported the treatment of heparin with periodate followed by partial acid hydrolysis which results in fragmentation of the chains and partial destruction of the functional groups.
Another example of a non-anticoagulant heparin is described by Casu, B. et al., Arzneim Forsch/Drug Res (1986) 36:637-642. They studied the effect of periodate oxidation on the anti-lipemic (lipoprotein lipase-releasing) activity of heparin. In this study, the heparin was oxidized with periodate and the products were reduced with borohydride. Although the authors stated that the product has the same molecular weight as the starting material, it is apparent from the figures presented in the paper that there is significant depolymerization.
PCT/SE92/00243 shows a non-anticoagulant heparin that has a molecular weight larger than the heparin starting material, and that is produced by periodate oxidation, partial depolymerization by alkali, and subsequent borohydride reduction.
PCT WO/92/17188, published Oct. 15, 1992 describes N-deacetylated heparinoids prepared by treating heparin with a reagent to effect N-deacetylation, then with periodate under conditions to effect complete conversion of vicinal-diols and vicinal OH/NH2 to aldehydes, and then reduction of the aldehydes to alcohols under conditions wherein fragmentation is prevented.
PCT WO/92/17187, published Oct. 15, 1992 describes NAC heparinoids prepared by oxidation of heparin/heparin sulfate with periodate to convert diols to dialdehydes and reduction of the resulting aldehydes all under conditions wherein fragmentation is prevented.
U.S. Pat. No. 4,847,338 describes certain heparin oligosaccharides with diminished anticoagulant activity compared to heparin produced by the depolymerization of heparin with heparinase. Although no data are presented, such fragments are stated to be useful for the treatment of septic shock and immune disorders.
The 2-O desulfated heparin compositions described by Jaseja, M., et al., in Can. J. Chem. (1989) 67:1449-1456, have non-anticoagulant activity.
Of the NAC heparins described above only fragments generated from heparin, either by periodate oxidation followed by base depolymerization or depolymerization with heparinase, have been suggested to be therapeutically useful for the treatment of shock or shock related syndromes. See U.S. Pat. Nos. 4,916,219 and 4,990,502. No data are presented, however, that actually show that these compositions have such activity. That such compositions might be useful to treat shock is premised solely on their anti-complement activity. No correlation was shown with this activity and beneficial effects for treating shock or shock related syndromes. It is worth noting that inhibition of complement activation prevents the formation of the complement fragments C5a and C3a. These fragments are one of at least a dozen different mediators thought to be involved in shock. To date the mere inhibition of the formation of C5a and C3a has not be shown to be beneficial for the treatment of septic shock. Moreover, there is no description of using the compositions for treating hypovolemic shock.
Unlike the reports described above, to date there are no reports on NAC heparin compositions consisting of substantially undepolymerized polymer produced by chemical modification of heparin that are useful for the treatment of hypovolemic shock or shock related syndromes. Furthermore, there are no reports on the types of NAC heparin fragments that would be useful for such treatment that result from the depolymerization of chemically modified heparin, or that are produced by direct chemical modification of hepafin fragments.
It will be appreciated that the availability of additional NAC heparin compositions that can be bencficially applied to treating hypovolemic shock or shock related syndromes will afford the physician a wider range of drugs to treat these diseases.